PPARγ基因修饰对兔骨髓间充质干细胞成脂分化及ADRP、LPL表达的影响

Effect of PPARγ Transfection on Differentiation of Rabbit MSCs into Adipocytes and Expression of ADRP and LPL

目的观察过氧化物酶体增殖物激活受体γ(PPARγ)基因转染对兔骨髓间充质干细胞(MSCs)向脂肪细胞分化的影响,探讨PPARγ在脂肪细胞早期分化中可能的调控机制。方法原代培养新西兰大白兔MSCs,应用脂质体介导法将pEGFP-N1-PPARγ表达载体转入MSCs中,G418筛选。分成不诱导组、空转染诱导组及转染诱导组以成脂诱导剂诱导分化,每日观察细胞形态学变化,镜下计数并计算脂肪细胞分化率,采用RT-PCR方法检测脂肪分化相关蛋白(ADRP)、脂蛋白脂酶(LPL)mRNA表达,Western blot检测ADRP、LPL蛋白表达。结果不诱导组细胞内没有脂滴出现,空转染诱导组和转染诱导组部分细胞成功分化为脂肪细胞,转染诱导组分化率明显高于空转染诱导组(P<0.05);不诱导组ADRP、LPLmRNA及蛋白均不表达,转染诱导组ADRP、LPLmR-NA及蛋白表达水平显著高于空转染诱导组(P<0.05)。结论PPARγ基因转染MSCs可促进其向脂肪细胞分化,增强其分化能力,可能与促进ADRP、LPL基因和蛋白的表达有关。

Objective To observe the effect of peroxisome proliferator - activated receptor γ（PPARγ） transfection on differentiation of rabbit marrow stromal cells（MSCs） into adipocytes, and to explore its possible regulatory mechanism during adipocytes early differentiation. Methods The MSCs of the New Zealand rabbit were cultured which had been put in the expression vector of pEGFP - N1 - PPARγ with the method of lipoplast, then alternated by G418, acquired stable trans-fection of MSCs. Adipocytic differentiation was induced by inducers for 14 days and was divided into three groups ： uninduced group, untransfected group and transfected group. The morphological changes of cells were observed daily and adipocytes were calculated under microscope. RT - PCR was employed to detect the expression levels of ADRP and LPLmRNA. Western Blot was used to observe the expression of ADRP and LPL protein. Results There was no adipocytes in uninduced group. MSCs differentiated into adipocytes untransfected group and transfected group. The rate of adipocytes in transfected group were markedly higher than those in untransfected group （ P 〈 0.05 ）. There were no expression of ADRP and LPL in uninduced group. The expression levels of ADRP and LPL mRNA and protein in transfected group were increased significantly compared with untransfected group （P〈0.05）. Conclutions PPARγ genetic transfection can reinforce the ability of MSCs differentiating into adipocytes, which may be related to the increased expression of ADRP and LPL.